Familial amyotrophic lateral sclerosis (FALS) is an inherited disorder of motor neurons, which is associated with point mutations in the coding regions in the Cu,Zn-superoxide dismutase gene. Some transgenic mice were produced with several FALS mutants. Mice from one of the G93A transgenic lines that expressed mutant SOD in brain was reported to develop a stereotyped syndrome of motor neuron disease. The fact that the symptoms occurred against a background of normal mouse Cu,Zn-SOD activity suggests that dominant, gain-of-function mutations in SOD play key roles in the pathogenesis of FALS. We investigated the nature of this gain-of-function of FALS mutants. We previously reported that Cu,Zn-SOD has the free radical-generating function in addition to the normal dismutation activity. To compare these two enzyme functions of mutant and wild-type enzymes, we cloned wild-type and two mutant (G93A and A4V) cDNA of human Cu,Zn-SOD, overexpressed them in insect cells (sf9), purified proteins, and studied their enzymic activities for catalyzing the dismutation of superoxide anions and the generation of free radicals with hydrogen peroxide as substrate. Our results showed that wild-type, G93A, and A4V enzymes have identical dismutation activity. However, the free radical-generating function of the G93A and A4V mutants, as measured by the spin trapping and EPR methods, is enhanced relative to that of the wild-type enzyme (wild-type<G93A<A4V), particularly at lower hydrogen peroxide concentrations. This is due to a decrease in the values of Km for hydrogen peroxide for the G93A and A4V mutants (wild-type>G93A>A4V), while k(cat) is identical for these enzymes. Thus, the ALS symptoms are not caused by the reduction of Cu,Zn-SOD dismutation activity with the mutant enzymes; rather, it is induced by a gain-of-function, an enhancement of free radical-generating function. In addition, our finding that A4V mutant has the lowest Km value for hydrogen peroxide is in accord with the clinical observation that A4V patients have the most aggressive form of FALS. Therefore, we suggest that the values of Km for hydrogen peroxide are associated with the rapidity of progress of the disease.